1. Field of the Invention
When a procedure is devised for determining the presence of a sample constituent--be the devised procedure gravimetric, volumetric, spectrophotometric or whatever mode--its efficacy in producing reliable results must somehow be assessed. Otherwise, the data developed is meaningless. Hence, devising an analytical procedure extends far beyond building a machine, formulating reagents or developing a technique. It also must of necessity include evaluating experimental error. There must be a way of predicting the dependability of the data produced by the procedure.
The easiest, most direct way to study parameters such as reproducibility, sensitivity, accuracy and need for calibration is to subject the procedure to a test sample wherein the analyte presence and/or concentration is known beforehand, i.e., a control solution. The data furnished by the procedure can then be compared with known data and any discrepancies properly noted.
The present invention concerns itself with the assessment of procedures for determining the presence and/or concentration of bilirubin in a liquid sample. Moreover, it relates to a device for preparing a bilirubin control solution for use in assessing the performance of various bilirubin determination procedures.
The chemistry and biology of the bile pigments are quite complicated; some of the steps in the metabolic pathways being still shrouded in mystery. Much of the older literature on the subject is now obsolete, and not of primary concern to the clinical chemist. Bilirubin, however, is one of the bile pigments occurring somewhat early in the metabolism of heme, and substantial analytical literature is available.
Bilirubin originates primarily from the breakdown of the heme moiety of hemoglobin in senescent erythrocytes by the reticulo-endothelial system. This occurs primarily in the spleen, liver and bone marrow.
Bilirubin which is formed from the breakdown of hemoglobin is transported in the plasma bound to a carrier such as albumin or, in small amounts, .alpha.-globulins and other serum proteins. Anionic drugs, such as salicylates and sulfa, or other anions, such as free fatty acids, can compete for these binding sites and substantially reduce the bilirubin transport capacity of the plasma. It is hypothesized that bilirubin dissociates from its carrier protein in the liver cell membrane, and it is transported intracellularly by some act or process, either unbound or attached to high-affinity, specific acceptor systems.
Conjugation of bilirubin with glucuronic acid, and, to a lesser extent with sulfuric and possibly other acids, occurs in the liver. Conjugated bilirubin is excreted from the liver cell into the bile canaliculus. In the intestinal tract a small fraction of the conjugated bilirubin excreted in the bile is hydrolyzed and the unconjugated bilirubin reabsorbed. Consequently, practically all excreted bilirubin is in the conjugated form.
The diagnostic value of determining bilirubin in urine has long been recognized. Normal urinary bilirubin levels are less than about 0.05 milligrams per deciliter (mg%). Accordingly, elevated bilirubin levels in urine connote the possible existence of a pathological condition in a patient. For example, high bilirubin levels can result from biliary obstruction, and hemolytic and hepatic disease. It is generally recognized that the presence of bilirubin in urine at concentrations greater than the abovementioned 0.05 mg% indicates the desirability of performing more comprehensive diagnostic procedures determinative of the specific pathological condition causing the elevated urinary bilirubin concentration.
As stated supra, essentially all bilirubin appearing in pathological urines or other bodily excreta is in the glucuronate conjugated form. Many analytical systems exist in the art for determining this form of bilirubin.
2. Description of the Prior Art
Over 70 tests have been proposed for the qualitative determination of bilirubin in urine. In general, these can be grouped into four categories depending on the principle used: (a) observation of the color of the urine sample; (b) the titration of the urine sample with a dye (e.g. methylene blue) until the dye color dominates over the bilirubin color; (c) oxidation of bilirubin to characteristic colors; and (d) diazo-coupling. Moreover, there are commercially available dip-and-read reagent strips on the market, such as those available from the Ames Division of Miles Laboratories, Inc., known as BILI-LABSTIX.RTM., ICTOSTIX.RTM., MULTISTIX.RTM., and N-MULTISTIX.RTM., as well as ICTOTEST.RTM. reagent tablets, also available from Ames Division.
Bilirubin is conventionally determined in routine urinalysis based on its reaction with various diazonium compounds in an acidic medium to form a colored azobilirubin complex. While several test formats are reported in the literature, the most commonly used test format in the clinical laboratory is that generally referred to as a test strip. The diazonium compound is incorporated in a carrier capable of absorbing a predetermined amount of urine when dipped momentarily into a urine sample. Any resulting colorimetric response may be read in less than one minute. Such test strips as those Ames Division products exemplified above utilize these principles.
The preparation and use of a bilirubin test strip is described in detail in U.S. Pat. No. 3,585,001. While the test strips which have been described in the art provide very rapid and convenient means for detecting urinary bilirubin, it is generally known that the available test strips are not sufficiently sensitive to detect levels of bilirubin only slightly elevated from the normal level, i.e., between 0.2 and 0.4 milligram (mg) bilirubin per 100 milliliter (ml).
There have been a few reported attempts to increase the sensitivity of the reaction between diazonium compounds and urinary bilirubin; however, the test systems that have resulted have certain disadvantages. U.S. Pat. No. 3,880,588 describes a class of diazonium compounds designed to enhance the colorimetric response of the azobilirubin complex and to decrease interfering color reactions with urobilinogen, which is structurally and chemically very similar to bilirubin. The described diazonium compounds, unlike the conventional compounds, form interfering colored products with such constituents of urine as homogentisic acid and 5-hydroxyindole-3-acetic acid. The latter is a normal constituent of urine and as little as 1 mg% of such constituent in urine causes false positive results using the diazonium compounds described in this patent.
Another attempt to increase the sensitivity of the test strip-incorporated diazonium reagents is described in U.S. Pat. No. 3,853,476 which discloses the use of certain phosphoric acid diesters as sensitizing or potentiating agents for the reaction between the diazonium compound and bilirubin. However, due to the incompatibility between the phosphoric acid diesters and aqueous media, test strips prepared according to this patent must be manufactured by a double-impregnation process.
It should be mentioned that various so-called "accelerating agents" have been described in the art relative to the detection of bilirubin in serum by the diazo-coupling reaction. Such agents have included caffeine, dyphylline, sodium acetate, sodium benzoate, gum arabic, and various other chemically unrelated compounds.
The use of such accelerating agents in serum bilirubin tests was described in the literature as early as the 1920's but has never been applied in general to urinary bilirubin tests. This has been due to the generally recognized fact that such accelerating agents act on a form of bilirubin that is not present in significant amounts in urine. Such accelerators are reported to promote the diazo-coupling of free bilirubin.
However, no effect on the coupling of conjugated bilirubin has been reported, since in serum bilirubin tests the conjugated forms of bilirubin react relatively rapidly with the diazonium compounds without the need for accelerators. Hence, the conjugated forms of bilirubin in serum are referred to as direct-reacting bilirubin, whereas free bilirubin, which requires the presence of an accelerator in order to react rapidly, is referred to as indirect-reacting bilirubin. The fact that the scientific community views the effect of the reported accelerating agents as being restricted to the diazo-coupling reaction of free bilirubin, and not applicable to the reaction with conjugated bilirubin, is well supported by the concurrence of the primary review publications considered authorative in the art. For example, reference may be made to Henry, R. J., Clinical Chemistry, Principles and Technics, Harper and Row (1964) pp. 577-583; With, T. K., Bile Pigments, Academic Press (1968) pp. 324-327; and the Journal of American Medical Technologists, volume 31 (1969) pp. 707-710.
A single investigator has reported the use of dyphylline in a urinary bilirubin determination--Scandanavian Journal of Clinical Laboratory Investigation, supplement 56 (1961). However, such use was specifically designed to accomplish the same effect as discussed in the literature relative to serum bilirubin tests, namely, to accelerate the diazo-coupling of free bilirubin which, as is known, could only be present in the urine tested in very small amounts. The described procedure involves a cumbersome liquid test system, and there is no suggestion of a rapid and sensitive test strip system. Moreover, the described procedure has received little attention from those skilled in the art in their development of more sensitive bilirubin test strips, as is evidenced by their resort to the disadvantageous test systems disclosed in the previously discussed U.S. Pat. Nos. 3,853,476 and 3,880,588.
The present invention relates to a way of assessing both the reliability of the bilirubin-sensitive procedure selected, as well as the proficiency of the technician in assessing the results. This is achieved through the use of a reference or control sample--a test sample in which the chemical composition and physical characteristics simulate the test samples to be analyzed.
Exemplary of a commercially available control is CHEK-STIX.RTM., marketed by Ames Division, Miles Laboratories, Inc., which utilizes unconjugated bilirubin present in a paper pad at the end of a plastic strip. When the strip is immersed in a predetermined volume of water for a predetermined time, a control solution results having a calibrated bilirubin concentration. The bilirubin used for this purpose is in the unconjugated form, and thus does not produce the same color as a pathological specimen containing bilirubin glucuronate conjugate. The present invention concerns itself with solving this problem. Thus, it provides a control solution, using unconjugated bilirubin, yet which does not provide atypical results in reagent systems devised to respond to pathological urine specimens containing bilirubin.